1. Field of the Invention
The present invention relates to a DNA sequence, a polypeptide encoded by this sequence, and to the use of said DNA sequence and polypeptide in the production of amorphadiene.
2. Description of the Related Art
Human malaria is a commonly occurring widespread infectious disease, caused in 85% of the cases by Plasmodium falciparum. This parasite is responsible for the most lethal form of malaria, malaria tropicana. Each year, malaria causes clinical illness, often very severe, in over 100 million people of which eventually over 1 million individuals will die. Approximately 40% of the world's population is at risk of malaria infection (as estimated by the World Health Organization).
Malaria has traditionally been treated with quinolines, such as quinine, chloroquine, mefloquine and primaquine, and with antifolates. Unfortunately, most P.falciparum strains have become resistant to chloroquine, and some have developed resistance to mefloquine and halofantrine as well. Thus, novel antimalarial drugs to which resistant parasites are sensitive are urgently needed. Artemisinin, as well as its semisynthetic derivatives are promising candidates here.
Artemisinin (FIG. 1), [3R-(3α,5aβ,6β,8aβ, 9α,12β,12aR*)]-Octahydro-3,6,9-trimethyl-3,12-epoxy-12H-pyrano[4,3-j]-1,2-benzodioxepin-10(3H)-one; molecular weight 282.35), also called arteannuin, qinghaosu or QHS, is a sesquiterpene lactone endoperoxide isolated from the aerial parts of the plant Artemisia annua L.
Artemisia annua L., also known as quinghao (Chinese), annual or sweet wormwood, or sweet annie is an annual herb native to Asia. A.annua, a member of the Asteraceae, belongs to the tribe Anthemideae of the Asteroideae, and is a large herb often reaching more than 2.0 m in height. It is usually single-stemmed with alternating branches. The aromatic leaves are deeply dissected and range from 2.5 to 5 cm in length. Artemisinin is mainly produced in the leaves as a secondary metabolite at a concentration of 0.01-0.6% on a dry weight base in natural populations. Artemisinin is unique to the plant A.annua with one possible exception of A.apiacea L. The A.annua used in this invention is of Vietnamese origin.
Because of its low concentration in plants, artemisinin is a relatively expensive resource for a drug. Current research has thus been aimed at producing artemisinin at a larger scale by organic synthesis. However, because artemisinin consist of seven chiral carbon atoms, theoretically 27=128 isomers can be formed of which only one is identical-to artemisinin. Because of this complex structure of artemisinin, production of this compound by organic synthesis is not profitable from a commercial point of view.
Genetic engineering of the biosynthetic pathway of artemisinin may give rise to higher artemisinin levels in plants. To be able to interfere in the biosynthesis of artemisinin, the biosynthetic pathway has to be known, either completely or partially. Several attempts to elucidate the entire biosynthetic pathway have been undertaken. Until now, however, the exact pathway has remained largely unknown.
In the research that led to the present invention, a unique pathway has been discovered which has not been published before. This pathway involves inter alia the formation of the artemisinin precursors amorpha-4,11-diene (1β,6β,7β,10αH-amorpha-4,11-diene) and the hydroperoxide of dihydroarteannuic acid. These precursors that were found in A.annua have not been described before in literature.
From literature it is known that terpene cyclases (synthases) are branch point enzymes, which likely play an important role in terpenoid biosynthesis. The working hypothesis for this invention is thus that over-expression of such a branch point enzyme (terpene cyclase) may increase terpenoid production in an organism. Factors that may influence the success of such an approach are, in the case of artemisinin, the number and nature of the subsequent biosynthetic steps leading to artemisinin. FIG. 2 shows the biosynthetic pathway of artemisinin as postulated by the present inventors. This pathway is divided into three parts:
The first part (Part I) represents the terpenoid (Isoprenoid) pathway. This pathway is a general pathway. Farnesyl diphosphate (farnesyl pyrophosphate) (FPP), for example, is present in every living organism and it is the precursor of a large number of primary and secondary metabolites. It has been established that FPP is the precursor of all sesquiterpenes. Thus, by definition FPP is the precursor of artemisinin.
Part II displays the cyclization of the general precursor FPP into the highly specific precursor amorpha-4,11-diene (also referred to as amorphadiene), the first specific precursor of artemisinin. In this pathway amorphadiene synthase is a branch point enzyme, having a key position in the biosynthetic pathway of artemisinin.
In part III, dihydroarteannuic acid (DHAA), also called dihydroartemisinic acid, is photo-oxidatively converted into its hydroperoxide (DHAA-OOH). This hydroperoxide of DHAA will spontaneously oxidize into artemisinin. No enzymes are involved in this part of the pathway and therefore it is impossible to alter artemisinin production by over-expression of genes involved in this part of the pathway.
Cytochrome P-450 catalyzed enzymes and an enoate reductase are probably involved in the conversion of amorphadiene into DHAA, the transition state between part II and part III (see FIG. 3). Because no intermediates of this part of the pathway are known or present (accumulated) in detectable amounts, in the plant, (except arteannuic acid, also called artemisinic acid or 4,11(13)-amorphadien-12-oic acid) it is likely that these precursors are very rapidly converted into DHAA. A rate limiting step in this part of the pathway is not very likely.
Taking all these aspects into account the inventors concluded that the most logical step to be altered by genetic interfering, is the conversion (cyclization) of FPP into amorphadiene by amorphadiene synthase.
The object of the present invention is therefore to provide a way in which artemisinin can be obtained via an at least partially biological route.
This object is achieved by the provision of a DNA sequence which exhibits at least a 70% homology to the sequence as shown in FIG. 12, and which codes for a polypeptide having the biological activity of the enzyme amorphadiene synthase.
The biological activity of the enzyme amorphadiene synthase relates to the conversion of the general precursor farnesyl pyrophosphate (FPP) into the specific artemisinin precursor amorpha-4,11-diene, which, in A.annua, is further converted to artemisinin. Suitable genes according to the invention can be selected by testing the expression product of the gene for its ability to convert FPP into amorpha-4,11-diene.
By transforming a suitable host cell with the DNA sequence of the invention, the conversion of farnesyl pyrophosphate (FPP) into the highly specific precursor amorphadiene can be increased or induced if this conversion route is not naturally present in the organism. In the latter case, the organism should comprise or be able to produce FPP. Suitable host cells are for example bacterial cells, such as E.coli, yeast cells like Saccharomyces cerevisiae or Pichia pastoris and in particular oleaginous yeasts, like Yarrowia lipolytica, or plant cells such as those of A.annua. 
Several plants are capable of producing large amounts of FPP making them potential organisms for amorphadiene production.
The potential oleaginous yeast host cells, like, for example, Yarrowia lipolytica and Cryptococcus curvatus, have the capacity to accumulate up to about 50%. (dry weight) of storage carbohydrates in oil bodies, making them very interesting candidates as production organisms for large quantities of terpenes. According to the invention, a way to obtain high levels of terpene accumulation is for example by means of re-direction of the metabolic flux in favor of the formation of amorpha-4,11-diene.
In analogy to the approach of an increased carotenoid production by the food yeast Candida utilis through metabolic engineering of the isoprenoid pathway as done by Shimada et al. (Appl. Environ. Microbiol. 64, 2676-2680 (1998)) the target genes according to the invention are acetyl CoA carboxylase (ACC, disruption), hydroxy-methyl-glutaryl CoA reductase (HMGR, over-expression), and squalene synthase (SQS, disruption) to obtain an increase of the precursor supplies, and amorpha-4,11-diene synthase over-expression to obtain accumulation of amorphadiene in such yeast cells. Because several expression systems (for example Muller et al., Yeast 14, 1267-1283 (1998); Park et al., The Journal of Biological Chemistry 272, 6876-6881 (1997); Tharaud et al., Gene 121, 111-119 (1992)) and transformation systems (for example Chen et al., Appl. Microbiol. Biotechnol. 48, 232-235 (1997)) are known for Y.lipolytica in literature, transformation and expression of the previously mentioned target genes in Y.lipolytica is possible without serious technical problems.
By adding FPP to a culture medium further comprising the enzyme of the invention (isolated as described in example 1), or transformed cells, e.g. E.coli, comprising the DNA sequence of the invention (as described in examples 3 and 4), which is expressed, FPP is converted into amorphadiene. Amorphadiene can then be used as a starting material for the production of artemisinin.
Transformed cells in which amorphadiene is produced as a result of the expression of amorphadiene synthase of the invention can be used either in disrupted form, by for example sonication, or as intact cells, as a source of amorphadiene.
Over-expression of the amorphadiene synthase encoding gene in A.annua will increase artemisinin production, because the terpene cyclase is expected to be the rate limiting step.
The results of the present research (postulated biosynthetic pathway of artemisinin) make the presence of a single major rate limiting step at the place of the amorphadiene synthase clear. Over-expression of the amorphadiene synthase encoding gene can increase the production of artemisinin in A.annua. 
The chemical structure of the first specific precursor of artemisinin, a cyclization product of FPP, was not known in literature. Neither has anyone so far detected such a compound in A.annua. Nevertheless it was possible to predict a likely structure for this cyclization product, based on the structure of DHAA and arteannuic acid (FIG. 3). The structure predicted in this way was consistent with a compound which is known in literature as 4,11-amorphadiene (J. D. Connelly & R. A. Hill in: Dictionary of terpenoids, Chapmann and Hill, London, England), as depicted in FIG. 4. This compound, isolated from Viguiera oblonqifolia, has previously been described by Bohlmann et al. under the incorrect name cadina-4,11-diene (Phytochemistry 23(5) 1183-1184 (1984)). Starting from arteannuic acid (isolated from A.annua), it was possible to synthesize amorphadiene. Amorphadiene obtained in this way was in all chemical and physical aspects identical to amorphadiene as described by Bohlmann et al., and this standard was used to show the presence of amorphadiene in a terpene extract of A.annua. 
A further object of the present invention is to provide a polypeptide having the biological activity of the enzyme amorphadiene synthase, obtainable by a process as described in example 1. This polypeptide can be used to convert FPP into amorphadiene which subsequently can be converted into artemisinin. Conversion can take place either in planta, when the polypeptide amorphadiene synthase is expressed in a plant that contains the necessary enzymes to further convert amorphadiene into artemisinin, or in vitro when FPP and the polypeptide (either in isolated form or as an expression product in a cell) are brought together in an incubation mixture.
Amorphadiene, produced by a suitable host organism transformed with the DNA sequence of the invention as precursor, can subsequently be chemically converted to dihydroarteannuic acid. Dihydroarteannuic acid per se can be used or in the production of artemisinin.
The chemical conversion of amorphadiene into dihydroarteannuic acid (FIG. 15) starts with the enantio-, stereo- and regioselective (anti-markownikoff) hydroboration of amorphadiene with BH3, yielding a trialkylborane, followed by an oxidation of the trialkylborane with NaOH/H2O2 yielding the alcohol (Advanced Organic Chemistry, Jerry March, 4th Edition, Wiley, 1992). A mild oxidation of the alcohol to the acid can be obtained by PDC (pyridinium dichromate) without attacking the second double bond (FIG. 15) (Organic Synthesis, M. B. Smith, 1st Edition, McGraw-Hill, 1994).
Many genes encoding enzymes involved in the biosynthetic pathway of farnesyl diphosphate are cloned and known in literature. For A.annua, for example, the sequence of the farnesyl diphosphate synthase encoding gene is known in literature (Y. Matsushita, W-K. Kang and V. Charlwood Gene, 172 (1996) 207-209). A further approach to introduce or increase the amorphadiene production in an organism, is to transform such an organism (for example A.annua) simultaneously with the DNA sequence of the invention with one or more genes involved in the biosynthesis of farnesyl diphosphate. The expression of a fusion protein of amorphadiene synthase and farnesyl diphosphate synthase may be an example here.
(Sesqui)terpenes, such as amorphadiene, are also known as flavor and fragrance compounds in the food and perfume industry. In addition, terpenes play a role in plant-insect interactions, such as the attraction or repulsion of insects by plants. Furthermore, dihydro-arteannuic acid, which is an intermediate in the metabolic route from amorphadiene into artemisinin in A.annua, can be used as an antioxidant.
Amorphadiene, obtained by (over)expression of the DNA sequence of the invention, or by using the polypeptide (amorphadiene synthase) of the invention, can be applied for these purposes as well.
The plants that can be used for this invention are preferably plants already producing artemisinin. A prime example is Artemisia annua, as this species contains the remainder of the pathway leading to artemisinin. However, this invention may also be used for the production of amorphadiene in plants, which, as mentioned before, can be used as a flavor or fragrance compound or biocide, or can be converted to artemisinin, either chemically or by bioconversion using microorganisms, yeasts or plant cells.
The plant that can be used for the production of amorphadiene is preferably a plant already producing sesquiterpenes, as these plants already have the basic pathway and storage compartments available, or a plant in which the biosynthesis of sesquiterpenoids can be induced by elicitation. The methods of this invention are readily applicable via conventional techniques to numerous plant species, including for example species from the genera Carum, Cichorium, Daucus, Juniperus, Chamomilla, Lactuca, Pogostemon and Vetiveria, and species of the inducible (by elicitation) sesquiterpenoid phytoalexin producing genera Capsicum, Gossyium, Lycopersicon, Nicotiana, Phleum, Solanum and Ulmus. However, also common agricultural crops like soybean, sunflower and rapeseed are interesting candidates here.